Project Summary Dilated cardiomyopathy (DCM) is a leading cause of heart failure. Genetic DCM, which runs in families, is now more commonly diagnosed, accounting for up to half of the reported cases. Despite the progress in unraveling the genetic basis of DCM, there is still a large gap in our understanding of the molecular events and signaling pathways that lead from a mutation to diverse disease phenotypes. The recent advent of new technologies, such as patient-specific induced pluripotent stem cells (iPSCs) and genome editing (CRISPR/Cas9), provide an unprecedented opportunity to study associations between genetic variability and disease susceptibility. By combining these breakthrough technologies, we are now poised to address one of the most critical issues in familial DCM, namely, the genotype-phenotype relationship from the ever-growing number of DCM-associated gene mutations. The overarching goal of our proposal is to utilize a multidisciplinary approach that integrates patient-specific iPSCs, genome editing, genetic screens, next generation sequencing technologies and transgenic animal models to gain novel insights into genotype-phenotype associations, and to dissect the molecular mechanism of genetic DCM pathogenesis. Our preliminary studies have implicated the activation of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) in the pathogenesis of DCM in a model of patient-specific iPSC-CMs. The central hypothesis of the revised proposal is that certain DCM mutations alter Ca2+ homeostasis and cause chronic ER stress, leading to prolonged activation of the UPR signaling. We will pursue three specific aims. In aim 1: we will establish an experimental platform to study the genotype- phenotype association of cardiac troponin T (TNNT2) and phospholamban (PLN) gene variants associated with DCM; in aim 2: we will decipher the role of ER stress and UPR activation in genetic DCM; and in aim 3: interrogate the mechanisms of DCM pathogenesis associated with TNNT2 mutations in vivo. We have assembled a multi-disciplinary team with extensive complementary and integrated expertise. We have provided compelling preliminary data to support the soundness of our hypothesis-driven research proposal, and we are well positioned to achieve the project goals within five years. If successful, our studies will provide a new paradigm for understanding the pathogenesis and treatment of familial DCM.